Photoinhibition of photosynthesis in vivo: Involvement of multiple sites in a photodamage process under CO 2- and O2-free conditions

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1 Biochimica et Biophysica Acta, 1142 (1993) Elsevier Science Publishers B.V. All rights reserved /93/$6. BBABIO Photoinhibition of photosynthesis in vivo: Involvement of multiple sites in a photodamage process under CO 2- and O2-free conditions Huashi Gong a, Stein Nilsen a and John F. Allen b a Department of Biology, University of Oslo, Blindern, Oslo (Norway) and b Plant Cell Biology, Lund Unicersity, Lund (Sweden) (Received 2 November 1992) Key words: Bicarbonate; D1 protein; Electron transport; Chlorophyll fluorescence; Photosystem I; Reaction center II; (L. gibba) Intact Lemna gibba plants were illuminated by photoinhibitory light in air, in air minus 2, in air minus CO 2, and in pure N 2. In pure N2, the degree of photoinhibition increased 3-5-times compared with that in air. This high degree of photoinhibition is described as photodamage. Photodamage was found to constitute a syndrome, that is, it is due to inactivation of multiple sites. These sites include RC II component(s) from P68 to QA; the Qa-site; and a component of PSI. In photodamage, the donor side of PS II and PS II excitation energy transfer remain unimpaired, but the size of the PS I antenna seems to decrease. Photodamage is distinguishable from photoinactivation. Photoinactivation occurred in air and could he attributed to inhibition of electron transport from QA to QB. During photoinactivation the D1 protein of RC II became degraded faster than the detectable inhibition of Qa reduction. The photoinhibition-induced rise in F occurred only during the process of photodamage but not during that of photoinactivation, and was a secondary event which arose as a consequence of photodamage. Atmospheric 2 alleviated photodamage but increased photoinactivation. The light-induced D1 degradation and inhibition of QA to QB electron transfer were enhanced in vivo not only by 2 but also by depletion of CO 2. Introduction Photoinhibition is a process by which photosynthetic rate is decreased as a result of exposure of oxygenevolving organisms to light intensities greater than those saturating photosynthesis. Photoinhibition consists of photoinactivation of reaction centre II and breakdown of D1 protein [1]. Based on experiments in vivo with green algae or cyanobacteria, and in vitro with thylakoid membranes or PS II particles, it is proposed that, during photoinactivation, the acceptor side of RC II is altered reversibly and electron flow from QA to QB is slowed down [1-5], followed by double reduction of QA, which is irreversible [5]. The inactivation of the acceptor side may induce changes in the donor side of Correspondence to: J.F. Allen, Plant Cell Biology, Lund University, Box 77, S-22 7 Lund, Sweden. Abbreviations: Asc, ascorbate; DI, QB-binding reaction centre protein of Photosystem I1; DCIP, 2,6-dichlorophenol indophenol; DPC, diphenylcarbazide; MV, methylviologen; PFD, photon flux density; PQ, plastoquinone; PS I, Photosystem I; PS II, Photosystem II; QA, the quinone which is the primary electron acceptor of PS II; Qa, the quinone which is the secondary electron acceptor of PS II; RC I, II, Photochemical reaction centre of Photosystem I, II. PS II [1,2]. Inactivation of the donor side results in formation of cation radicals at RC II which may further damage the RC II-D1 protein and accelerate photoinhibition [6-8]. Some reports have shown that PSI photochemical activity is also inactivated by photoinhibitory treatment in vitro in the absence of an added electron acceptor [9]. Photoinhibition of PSI requires either the presence of oxygen [9,1] or extremely reducing conditions [11,12]. The photoinhibition described above occurs in air. Photoinhibition can be strongly affected by composition of the gas phase. Photoinhibition is alleviated by the presence of CO 2 in vivo [13,14] or by addition of bicarbonate in vitro [15]. The role of oxygen is more complex. In presence of 2 the organism can dissipate a part of the excess energy through pseudocyclic electron flow [14,16] or through photorespiration [9,13]. Anaerobic conditions can greatly enhance susceptibility to photoinhibition [17,18]. On the other hand, the presence of 2 may be required for photoinhibition, since oxygen free-radicals formed by photosynthetic reduction of oxygen [19] may be responsible for breakdown of the RC II-D1 protein [2,21]. Some authors have suggested that photoinhibition has different

2 116 mechanisms under anaerobic and aerobic conditions [17,22]. The anaerobic conditions gave an increase in F and aerobic condition gave an decrease in Fv in isolated chloroplasts [17,22]. However, the differcnt sites of in activation in each case are still unclear. In the present work we find involvement of multiple sites during a photodamage process in vivo. The photodamage occurs under conditions of depletion of 2 and CO 2 which are similar to the anaerobic conditions reported earlier [15,17,18]. The results serve to distinguish photodamage from the photoinactivation which occurs in air. Further features of photodamage are described. Materials and Methods Plant material Axenic cultures of Lemna gibba L. G 3 were precultured as previously described [23]. The plants were then grown for 6-9 days at a PFD of 25 ~mol m -2 s-~ at 22 C. Plants at the 4-frond stage were selected for the experiments. Photoinhibitory treatment and photosynthesis measurement Plants were photoinhibited using tungsten-halogen lamps, at PFDs specified in results. The gas phases were controlled by flushing the reaction chamber with: air; 99.99% N2; N 2 plus 36 ppm CO 2 (equivalent to air minus 2); air minus CO 2. CO2 was removed from air by pumping it through a column filled with Carbosorb (BDH Chemicals, UK). Net photosynthetic rate was measured as CO 2 uptake using an infra-red gas analyser (Uras 2G) in a closed gas exchange system as previously described [24,25]. Thylakoid preparation Immediately after the photoinhibitory treatment, plants were homogenised with cold homogenisation medium consisting of sorbitol (.33 M), NaCI (1 mm), MgCi 2 (5 mm), EDTA (2 mm) and Tricine (5 mm) adjusted to ph 7.6 with KOH. The homogenate was filtered through four layers of fine nylon mesh directly into a centrifuge tube. 1/4 volume of the homogenisation medium containing sucrose (.33 M) instead of sorbitol was smoothly injected into bottom of the tube to form two clear phases of liquid. The mixture was centrifuged at 1 g for 3 min. The procedure described above is based on that of Mills and Joy [26]. The pellet of chloroplasts was resuspended with the sorbitol-containing medium for further experiments. For photosynthetic electron transport measurements, the thylakoid suspension was used at once; for the 77 K fluorescence detection, the samples of suspension were frozen in liquid N 2 before the measurements; for the gel electrophoresis, the suspension was centrifuged at 1 xg for 1 min and the pellet of thylakoid membrane was resuspended with the Tricine buffer and preserved at - 8 C. Chlorophyll concentration was determined according to Arnon [27]. Measurements of electron transport To measure PS ll-dependent electron transport, thylakoid suspension was added to Tricine buffer containing 1/xM DCIP, to give a final chlorophyll concentration of 4/zg ml- 1. The kinetics of photochemical reduction of DCIP were measured spectrophotometrically using either H2 or DPC (1 ~M) as electron donors [28]. The actinic light was obtained by use of a 665 nm short-pass filter to favour excitation of PS II [28]. For the PSI electron transport measurement, 2 uptake was detected using Clark-type polarographic electrode as previously described [29]. Ascorbate (5 mm) with DCIP (.1 mm) was used as an electron donor to methyl viologen (5 tzm) in the presence of DCMU (1/xM) and NaCN (1 mm). Measurements of chlorophyll fluorescence For room temperature fluorescence measurements, a PAM system (Waltz, Effeltrich, Germany) with standard accessories (PAM 11, 12 and 13) and computer analysis was used. The fluorescence decay after a single turnover flash (given by the xenon flash lamp, XST-13, Waltz) was measured according to Schreiber [3]. The flash intensity is intended to be high enough for one charge separation at every reaction centre and short enough to prevent double hits. During the whole period of experiments, the Lemna plants floated on growth medium in a home-made aluminium cuvette in which temperature and gas compositions were controlled. Lights for adaptation and photoinhibition were supplied with Olympus ILK-4 and Schott KL15 lamps respectively. The plants were dark adapted for 1 min before the flash. 77 K fluorescence emission was measured using a Perkin-Elmer LS-5 luminescence spectrometer as described previously [31]. Thylakoid suspension (see above) with a final chlorophyll concentration 7. Izg ml- i was frozen in liquid N 2 for at least 1 min before the measurement. Excitation was at 435 nm. The fluorescence emission maxima at 692 nm (PS II) and 737 nm (PS I) were normalised to the 55 nm fluorescence maximum of fluorescein (sodium salt) [32], present at a final concentration of 1./~M. Pulse-chase and analysis of RCII-D1 protein During the pulse period, 2 plants were incubated for 2 min in 4 ml cultivating medium containing 143 #Ci [35S]methionine (> 1 Ci mmol-l, Amersham) under a PFD of 25 ~mol m -2 s-1 at 2 C. At the end

3 117 of the pulse, 1 plants were removed for thylakoid preparation. The other plants were rinsed and incubated for 6 rain in cultivating medium containing 1 mm non-radioactive methionine under various atmospheric and light conditions. Thylakoid membrane and the D1 protein were separated by LDS-electrophoresis and autoradiography as previously reported [25]. D1 was quantified densitometrically. Results After Lemna plants werc illuminated by high light for 9 min, the degree of photoinhibition measured by CO 2 uptake was much higher in the gas phases lacking CO 2 than in those containing CO 2 (Fig. 1). In pure N 2 the degree of photoinhibition was the highest (8-9%, Fig. 1) and the photoinhibition induced in this condition recovered very slowly compared with that induced by other stress conditions [17,25]. We describe the photoinhibition in N 2 as photodamage. In air the degree of photoinhibition was much lower than that in N 2 (Fig. 1) and is described as photoinactivation. At PFD of 28 p.moi m -2 s-l, 2 alleviated photodamage but increased photoinactivation (Fig. 1). Table I shows electron transport partial reactions in photoinhibited plants. In pure N 2, the electron transport from H2 to DCIP measured under actinic light favouring PS II was inhibited by 43% which was higher than the degree of inhibition in any other gas phase. This indicates that the photodamage process is located at PS II. A similar degree of inhibition of DCIP reduction was obtained using DPC as an electron donor, showing that the water-splitting complex in PS 1I remained unimpaired. The question remains: how can photodamage inhibit CO 2 uptake to a much greater t- O. m. m.~ t- r- t" [] 15 umol rn_2s_l [] 28 ~ mot m s.-.- 7J~7, air air air minus minus 2 C2 Fig. 1. Loss of CO 2 up take after photoinhibitory treatment. Plants were illuminated for 9 min under the PFD and atmospheric conditions as indicated. Vertical bars represent S.D. N2 TABLE I Localization of photoinactication and photodamage in the photosynthetic electron transport chain Thylakoids were prepared from plants illuminated for 9 min under PFD of 28 ttmol m -2 s -1 in the gas phases indicated. The 'Control' was at PFD 25 #mol m -2 s- i in air. DCIP reduction was measured under actinic light preferentially ab~rbed by PS I1. The rates of electron transport are expressed as a percentage of the control rate. The control rate was 268_+ 4 #mol DCIP (mg chlorophyll) I h i for the PS li-dependent reactions H2-,DCIP and DPC --* DCIP, 7_+ 12 #mol 2 (mg chlorophyll)- t h t for the PS II- and PS 1-dependent reaction H2--* MV, and 167_+ 15 ~,mol 2 (mg chlorophyll)- l h- 1 for the PS-l-dependent reaction Asc ~ MV. The low rate of PS I1 + PS I reaction is possibly due to coupling of ATP synthesis, since uncouplers were absent during the measurements. HL, measured under saturating light; LL, measured under unsaturating light (PFD of 3/zmol m-2 s-1). Control Photoinhibitory treatment air air minus 2 air minus CO2 N2 H2 -* DCIP DPC --' DCIP 112 ~ MV Asc ~ MV HL IlL HL HL LL I(X) I(X) (} extent (Fig. 1) than PS II electron transport (Table I)? One possibility is that additional sites besides that in PS II may be inhibited. To check this, PS I electron transport from reduced DCIP to methyl viologen was measured before and after 9 min photoinhibitory treatment. In N 2, the rate of PSI light-saturated electron transport decreased by 8% and the rate of PS I light-limited electron transport decreased by 27%, while in the other atmospheric conditions PSI was not significantly influenced during photoinhibition (Table I). This result indicates that a site in PSI between P7 and the Fe-S centres is inactivated during the photodamage process. This is in agreement with previous reports using isolated chloroplasts, which showed that under extremely reducing conditions a site between A and Fe-S X of PSI was photoinhibited [12]. To probe additional effects on PS I and PS II, we used 77 K fluorescence emission spectroscopy. We obtained 77 K fluorescence emission spectra of thylakoids isolated from Lernna plants before and after photoinhibition under the four atmospheric conditions described (Table II). Two emission maxima, at 692 and 737 nm, were observed in all cases. The fluorescence emission at 692 nm, which may reflect the state of the PS II core and recombination luminescence of the RC II [33], showed a pattern of changes (Table II) consistent with those in electron transport (Table I). Fluores-

4 118 cence emission of PS II was lowered by the CO2-free gas, and was lowered by 5% after photoinhibition in N 2 (Table II). Fluorescence emission at 737 nm, which may be ascribed to LHC I and the PSI core [34,35], decreased by 42% after photoinhibitory treatment in pure N 2 (Table II). The drastically decreased 77 K fluorescence emission of PS I again suggests that PSI may be inactivated during the photodamage process. The size of the PS I antenna may have decreased. This is supported by the decreased 77 K fluorescence emission at 737 nm (Table II) as well as by the inhibited PS I light-limited electron transport (Table I). 77 K fluo- rescence of PS II as well as PSI was relatively unaffected by photoinactivation, especially in the absence of 2 (Table II). Most previous investigations on the mechanism of photoinhibition have been carried out in air and the site of inactivation was located at PS II. Table I showed that in N2, PS II-dependent electron transport was inhibited 2.5-times more than that in air. This indicates that the photodamage brought about by CO 2- and O2-free conditions results either from more photoinactivated sites or from acceleration of the damage at a single site within PS II. To estimate the site of block- 4 ms : ~trol.!contro, PI, C F,,,o - :'~~Control "t 15 PI, A 15 +CAP Fig. 2. The fast phase of fluorescence decay was slowed down by presence of O 2, absence of CO 2, and addition of chloramphenicol during photoinhibition. Intact plants were given a single turnover flash before (Control) and after (PI) photoinhibition of 9 min in air (A), air minus 2 (C), air minus CO2 (O) and pure N 2 (N) under a PFD of 28 #molm 2 s- l or 15 #,mol m- 2 s- t in air and in the presence or absence of chloramphenicol (CAP) at a final concentration of 5 #,g ml - 1.

5 119 TABLE II 77 K fluorescence emission during photoinhibition Lemna plants were photoinhibited at PFD of 28 p.mol m -2 s -l for 9 min in the atmospheres indicated. Fluorescence spectra were obtained for thylakoid extracts at equal chlorophyll concentration (7 p.g ml-1), and were normalised to fluorescence at 55 nm from 1 ~tm fluorescein. The normalized values for total fluorescence emission are given in arbitrary units + S.D. The 'Control' condition was the same as that in Table I. Control Photoinhibition air air minus O z air minus CO 2 N2 PSII (F692) PSI (E737) E692/F (I.58 5: : : :.12.3(I 5: :..65 5: age within RC II during photoinhibition, intact plants were given a single turnover flash after QA and the PQ pool were oxidised in the dark. The kinetics of fluorescence decay following the flash (Fig. 2) represent QA reoxidation [3,36]. The fluorescence decay during 4 ms showed three exponential components (data not shown). The first component reflects the forward electron flow from QA to QB [36,37] and the tl/2 of this phase is shown in Table III. The fast phase was slowed down by increasing PFDs (Fig. 2 and Table III). After 9 min illumination at PFD of 28/xmol m -2 s-1, the tl/2 of the fast phase increased in all gas phases. The increase of t~/2 of the fast phase was 2% greater in the presence of 2 than in absence of 2, and 19% greater in the absence of CO 2 than in presence of CO 2 (Fig. 2 and Table III). Photoinhibition in pure N 2 resulted in a similar degree of inhibition of QA to QB electron transport as that which occurred in air (Table III). In contrast, under PFD of 28,u.mol m -2 s -l, the inhibition of CO 2 uptake was almost 3-times higher in N 2 than in air (Fig. 1), and inhibition of PS II-dependent electron transport 2.5-times higher in N 2 than in air (Table I). The higher degree of photoinhibition of CO 2 uptake might be attributed to PSI inhibition. Thc higher inhibition of PS II electron transport in N 2 could be due to inhibition of RC II components from P68 to QA, since water oxidation itself seems to have been unimpaired (Table I) and since the inhibition of clectron flow from QA to QB was inhibited to a similar degree in N 2 as in air (Table lid. After photoinhibitory treatment, intrinsic fluorescence, F, incrcased in N 2 and, to a lesser extent, in air minus CO 2. F did not increase in the other gas phases (Table III). The increased F o can be due to RC II inactivation [38] by trapping of QA [3,4] or by Q~ destabilization [1,2]. F did not rise in air in vivo, although the QA reoxidation was inhibited (Fig. 2 and Table III). In addition, F does not rise in vitro when an electron acceptor such as ferricyanide is present [1]. Therefore the rise of F in N 2 indicates that QA oxidation is blocked due to the over-reduction of the PQ pool. The fluorescence kinetic studies (Fig. 3) show that, during photoinhibition in N 2, F started to rise after 2 min and came to a maximal level at 75 min, but in air F did not rise at any time (Fig. 3A). In N 2 the net photosynthetic rate was photoinhibited by over 5% even after an illumination for 1 min (Fig. 3B), while F did not change (Fig. 3A). This suggests that the rise in /7o is a secondary event arising as a consequence of photodamage. Moreover, the rise in F during photodamage may indicate that PS II charge separation is inactivated [38]. This is supported by the TABLE III Effect of photoinactit'ation and photodamage on QA reoxidation and RC H ac&,ity Fluorescence decay was recorded under the conditions described in Fig. 2. Yma~ was the maximal fluorescence following one flash. F and Ym~x values after photoinhibitory treatment are expressed as percentages of the values before photoinhibitory treatment. The 1% values for F o and for Ymax were the averages from the controls of each experiment. Photoinhibitory tl/2 (#s) of fast phase /7 5: S.D. (%) Ymax (%) condition control photo- increase photo- photoinhibited in t~/2 (%) inhibited inhibited 15 ~mol m -2 s -~ air air minus air minus CO ) l N z air + CAP ,o.mol m-2 s- air air minus air minus CO N

6 12 13 > 12 > '= 11 O IJL 1' 9 --~ 1 8O o '~-. 6 o ~ e- 4 i. o ~ o e- I--O- air j Time (min) 1 Fig. 3. Photodamage occurred earlier than the rise in initial fluorescence (F). Plants were photoinhibited under a PFD of 28 ~.mol m -2 s -) in air or pure N 2. F (A) and net photosynthetic rate (B) were measured at the times indicated and are expressed as percentages of photoinhibition (B). decrease in 77 K fluorescence emission at 692 nm (Table II), which is thought to be fluorescence from the photoactive pheophytin a that functions as an electron acceptor in RC II [33]. The lowered photosynthetic rate (Fig. 1) and PS II-dependent electron transport (Table I) in N 2 could partially result from impaired energy transfer from the TABLE IV Effect of increased actinic light intensity on rate of PS H electron transport after photomhibition The plants were photoinhibited in the same conditions as in Table I. PS 11 electron transport was measured in the actinic light as indicated. PFD used for PSII activity (H 2 ~ DCIP, txmol (mg Chl)- = h i measurement control photoinhibition air air minus 2 air minus CO 2 N 2 56 p.mol m 2 s-i tamol m -2 s"! antenna to the reaction centre of PS II. This interpretation would be consistent with the observed rise of F (Table 1II and Fig. 3) if the latter is caused by partial detachment of antenna pigments from the RCs [39]. In this case, the decreased photosynthetic rate in N 2 (Fig. 1 and Table I) could be reversed by increasing the actinic light intensity. To test this, we measured PS II electron transport under the actinic light intensities below and above saturation. Table IV shows that after the plants were illuminated by high light in N:, the degree of photoinhibition of electron transport could not be improved even if the measuring light intensity was increased by 5 times. This indicates that energy transfer from the antenna to the reaction centre of PS II is unlikely to have impaired in these experiments. Photoinactivation without photodamage occurred under high PFD in atmospheres containing CO 2. The A MW (kd) Gel Autoradiograph P C PC PC P C P C 24- B [] pulse chill degr~s.o "~ leo 4~ <Dll, A C N O Control Pl / 12 ~ 8 4o 'i li -- air I r N2 minus 2 PI sir minus c~ / 1 o Fig. 4. DI degradation in vivo was accelerated by presence of 2 and absence of CO 2. Plants were pulse-labeled with [35S]methionine for 2 min (P) and chased for 6 min (C) under PFD of 15 #mol m 2 s-' in air (A), air minus 2 (C), air minus CO 2 (O) and pure N 2 (N). D1 is quantified by means of a densitometric scan from Panel A, and the result is shown in Panel B. 'Relative amount of DI' refers to area of the peaks of DI obtained from the scan.

7 121 rate of CO 2 fixation (Fig. 1) and PS II activity (Tables I and II) decreased by a lesser degree during photoinactivation than during photodamage. Fluorescence relaxation following a single turnover flash (Fig. 2) showed that the rate of electron flow from QA to QB was inhibited by 26% as represented by the tl/2 after the plants were illuminated for 9 min under PFD of 28 #mol m -2 s -1 in air (Table III). This inhibition was largely lowered in air minus O z (Fig. 2 and Table III). These results indicated that the photoinactivation is 2 dependent and largely due to the inactivation of QB-site of the D1 protein. Pulse-chase experiments showed that during the photoinactivation period of only 6 min, the RC II D1 protein degraded in air by more than 4% (Fig. 4). Thus D1 degradation was more rapid than inhibition of electron transfer to QB (Table III). D1 degradation was accelerated by presence of O 2 and absence of CO 2 (Fig. 4). D1 degradation without loss of electron transfer to QB could result from efficient D1 synthesis and processing [2,4], since loss of QB reduction was increased by chloramphenicol which is an inhibitor of protein synthesis (Fig. 2 and Table III). Discussion Based on studies in vivo, our results indicate that multiple sites are involved in a photodamage process that is induced by light in CO 2- and O2-free atmospheres. The inactivated sites include the RC II components from P68 to QA, the Qa site, and a component of PSI. The donor side of PS II and excitation energy transfer within PS II seem to be unimpaired. We suggest that the photodamage results from the over-reducing condition and depletion of bicarbonate. Bicarbonate is essential for keeping the DI-D2 reaction centre functional. In the D1 protein, one of the HCO3-binding sites possibly locates at the pairs of Arg-257 and His-252 [41]. In our experiments, when pure N 2 was introduced into the chamber containing the plants, internal CO~ and O 2 could be extremely low. This may have caused partial removal of bicarbonate from its D1 binding site, since in Lemna the thylakoid has a relatively low ph under physiological condition, and this favours the dissociation of HCO 3 to CO 2 [41]. High light may promote the removal of bicarbonate, since high light drives CO 2 assimilation. The removal of bicarbonate results in inactivation of quinone reactions [42]. Moreover, it may also lead to inactivation of another component, as indicated in the present work, located between P68 and QA, due to disruption of the PS II complex after bicarbonate removal. We suggest that photodamage is a process distinct from photoinactivation. Photoinactivation occurs i~ air and its site of action is generally agreed to lie within PS II. At the early stage of photoinactivation in vivo, high light drives PS II charge separation at high rates and results in over-reduction of the PQ pool due to rate limitation of NADPH oxidation and CO 2 assimilation. When the PS II is gradually inactivated, the PQ pool becomes gradually oxidised due to a rate limitation of PS II electron flow [1]. A high turnover and high ratio of PQ/PQH 2 favour the breakdown of D1 [7,43]. In contrast, photodamage is favoured by reducing conditions and by absence of bicarbonate from the reaction centres, when the PQ pool is kept in a fully reduced state. F rises during photodamage but not during photoinactivation (Table III and Fig. 3). During photoinactivation, reducing conditions may be transient, and thus insufficient to cause a rise in F. Photoinactivation is strongly temperature-dependent and is enhanced at low temperatures [9,25,44]. In contrast, photodamage is relatively temperature-independent [9,25] but the following recovery strongly depends on the temperature used during photodamage [25]. High temperature during photodamage inhibits subsequent recovery [25]. Moreover, oxygen alleviates photodamage but aggravates photoinactivation (Fig. 1, 2 and Tables I, III). From this point of view the contradictory effects of 2 on photoinhibition may be explained. Under favourable conditions, photodamage does not occur [13] because a series of protective mechanisms may function [1]. Presence of atmospheric CO 2 enables carbon assimilation to use the excess energy produced under high light. In the presence of 2, the Mehler reaction [14], photorespiration [13], thylakoid energization [39,45] and operation of the zeaxanthin cycle [46] may enable the organism to dissipate the excess absorbed light energy. In these cases an apparent nonphotochemical fluorescence quenching may be observed [45]. Moreover, during photoinhibition in air PS I cyclic electron transport may be gradually activated, resulting in greater thylakoid energisation [47,48]. This may prevent further photodamage. In pure N 2 these protective mechanisms are minimised and the excess, undissipated energy may inactivate or damage multiple sites within the photosystems. It has been reported that light-dependent D1 degradation requires the presence of oxygen at the acceptor site [1,21]. A fast turnover and a high ratio of PQ1PQH 2 in the PQ pool promotes the breakdown of DI during photoinhibition [7,43]. In CO 2- and O2-free conditions, the rate of 2 evolution and the 2 concentration are extremely low, and the PO pool is over-reduced in high light. However, D1 is then degraded rapidly (Fig. 4) and QB reduction is inactivated. We suggest that the rapid degradation of D1 is a result of removal of bicarbonate from the HCO3-site of DI. One important function for this site is protonation of Q~- or QB [41]. The accelerated DI degradation and Oa-site inactivation (Table III and Fig. 4) may result from the pres-

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